Arne K Schierz, Giacomo Rößler, Jan Philipp Schneider, Stefan A Tschanz, Christopher Werlein, Danny D Jonigk, Julia Schipke, Christian Mühlfeld
{"title":"Distribution and volume of mitochondria in alveolar epithelial type 1 cells in infant and adult human lungs.","authors":"Arne K Schierz, Giacomo Rößler, Jan Philipp Schneider, Stefan A Tschanz, Christopher Werlein, Danny D Jonigk, Julia Schipke, Christian Mühlfeld","doi":"10.1007/s00418-024-02332-7","DOIUrl":"10.1007/s00418-024-02332-7","url":null,"abstract":"<p><p>Alveolar epithelial type I (AE1) cells with their wide spatial expansion form approximately 95% of the outer surface area of the air-blood barrier inside the lung. Serial block-face scanning electron microscopy (SBF-SEM) investigations led to the hypothesis that AE1 cell mitochondria are preferentially distributed as aggregates in those parts of AE1 cells that are located above connective tissue pillars between capillaries, thus not increasing the thickness of the diffusion distance for oxygen and carbon dioxide. Furthermore, it was hypothesised that postnatal development requires adapting the amount and distribution of mitochondria in AE1 cells. Human lung samples from three infant (26 and 30 days, 6 months) and three adult (20, 39 and 40 years) samples were investigated by light microscopy, transmission electron microscopy and stereology. The volume fraction of mitochondria was similar in infant and adult lungs with a mean value of 6.3%. The ratio between mitochondrial profiles on top of capillaries or above connective tissue pillars was approximately 3:1 in infants and adults. However, regarding the volume of both cytoplasmic compartments, infants showed a higher number of mitochondrial profiles on top of capillaries while adults showed a higher number above connective tissue pillars. Samples of three additional adult lungs were analysed by confocal laser scanning microscopy. Again, mitochondria were not preferentially found as aggregates above connective tissue pillars. In conclusion, AE1 cell mitochondria were not preferentially found as aggregates, showed the same volume density in infants and adults but differed in distribution between the age groups.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":"163 1","pages":"7"},"PeriodicalIF":2.1,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142667486","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Gamze Altun, Mehmet Emin Önger, Stefano Geuna, Abubaker El Elhaj, Stefania Raimondo, Ömür Gülsüm Deniz, Suleyman Kaplan
{"title":"The amelioration effects of ankaferd blood stopper, platelet gel, and Momordica charantia on peripheral nerve injury in the rats: a stereological and ultrastructural study.","authors":"Gamze Altun, Mehmet Emin Önger, Stefano Geuna, Abubaker El Elhaj, Stefania Raimondo, Ömür Gülsüm Deniz, Suleyman Kaplan","doi":"10.1007/s00418-024-02333-6","DOIUrl":"10.1007/s00418-024-02333-6","url":null,"abstract":"<p><p>Peripheral nerve injuries are commonly encountered in clinical practice. Peripheral nerve injuries most commonly result in serious problems affecting quality of life. The present study is designed to research the possible neuroprotective effects of Ankaferd blood stopper (ABS), platelet-rich plasma (PRP), and Momordica charantia (MC) on regeneration using unbiased stereological techniques. In total, 30 female 8-10 week Sprague Dawley rats were randomly divided into five equal groups; control (CG), the group exposed to sciatic nerve resection (Gap) (CGG), additionally following the surgical process ABS, MC, and PRP were injected into collagen tube (ABSG, MCG, PRPG). Nucleator and fractionator methods were used to estimate the number of myelinated and unmyelinated axons, axon area, and myelin sheath thickness. Also, an electron microscopic evaluation was performed. Regarding the number of myelinated axons, significant increases were found in the ABSG, MCG, and PRPG compared with CG (p < 0.05). The protective effects of ABS, PRP, and MC on peripheral nerve regeneration in experimental models in rats were shown using electrophysiological and stereological assessment parameters.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":"163 1","pages":"5"},"PeriodicalIF":2.1,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142667641","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alessandro Miraglia, Laura Giannotti, Francesco De Nuccio, Antonella Sonia Treglia, Michele Maffia, Dario Domenico Lofrumento, Bruno Di Jeso, Giuseppe Nicolardi
{"title":"Anaplastic thyroid carcinoma: vimentin segregates at the invasive front of tumors in a murine xenograft model.","authors":"Alessandro Miraglia, Laura Giannotti, Francesco De Nuccio, Antonella Sonia Treglia, Michele Maffia, Dario Domenico Lofrumento, Bruno Di Jeso, Giuseppe Nicolardi","doi":"10.1007/s00418-024-02329-2","DOIUrl":"10.1007/s00418-024-02329-2","url":null,"abstract":"<p><p>Anaplastic thyroid carcinoma (ATC) ranks among the most lethal human cancers. Increased migratory and invasive capabilities are critical in malignancy and are often secondary to epithelial-mesenchymal transition (EMT). However, it is not clear whether the invasive behavior of ATC is associated with the presence of EMT. In this study, we used a murine xenograft model (4-week-old male BALB/c NU/NU mice) with the human anaplastic cell line, FRO. We adopted an automated, eye-independent method to reconstruct the total/subtotal area of the tumors. To probe EMT, we evaluated the immunostaining of mesenchymal/epithelial markers at the front and center of the tumors. The transplanted cells invariably gave rise to tumor masses that histologically closely replicated patient tumors. The staining with hematoxylin-eosin and immunostaining with cytokeratin 18, an epithelial marker, were similar. However, the immunostaining of cytokeratin 18 versus vimentin, a mesenchymal marker, were strikingly dissimilar, since vimentin showed a staining concentrated at the front, rapidly declining towards the center of the tumor. The overlay, after color conversion, of cytokeratin and vimentin staining showed maximal coincidence at the front, which was rapidly lost towards the center. The results show EMT signs at the front of the ATC, which are probably at the basis of its tremendous invasiveness. Moreover, methodologically, an automated \"eye-independent\" acquisition of the total/subtotal area of the tumors drove the selection of second, high-magnification, automated field acquisition. Future studies may extend these results along the perspective of a personalized diagnostic procedure.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":"163 1","pages":"6"},"PeriodicalIF":2.1,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142667537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Novel role of curcumin as inhibitor of β-amyloid-induced lamin fragmentation.","authors":"Md Selim Hossain, Md Aminul Haque, Il-Seon Park","doi":"10.1007/s00418-024-02331-8","DOIUrl":"10.1007/s00418-024-02331-8","url":null,"abstract":"<p><p>Oligomer amyloid beta 42 (Aβ) is considered the key pathogenic molecule in Alzheimer disease (AD) and causes specific lamin fragmentation. Curcumin has been recognized for its protective effects against Aβ-induced toxicity in AD, though its underlying mechanism remains unclear. In this study, the inhibitory mechanism of curcumin against Aβ-induced lamin fragmentation and cell death was investigated. Human neuroblastoma cells were used to examine Aβ-induced lamin fragmentation and lamin deformation by immunoblotting and confocal microscopy, while cell viability was measured using MTT and alamarBlue assay. Caspase and cathepsin L activity were assessed by spectrofluorometry, and Aβ aggregation was evaluated by ThT assay. Our results demonstrated that curcumin inhibited Aβ aggregation, reducing intracellular Aβ uptake by 45% compared to Aβ-treated cells. Curcumin also inhibited the Aβ-induced intracellular calcium rise, subsequently leading to a onefold reduction in cathepsin L activity. This reduction in cathepsin L activity by curcumin blocked the Aβ-induced lamin fragmentation. Collectively, these findings suggest that curcumin inhibits Aβ-induced cell death by preventing Aβ entry and lamin cleavage, providing potential new insights for AD treatment.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":"163 1","pages":"2"},"PeriodicalIF":2.1,"publicationDate":"2024-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142619252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ana Vitoria Ferreira Dos Santos, Renan Gabriel da Silva Ferreira, Fernanda das Chagas Angelo Mendes Tenorio, Carina Scanoni Maia, Valdemiro Amaro da Silva Junior, Romildo de Albuquerque Nogueira, Bruno Mendes Tenorio
{"title":"Complex shape markers can detect alterations in the spatial distribution of cell nuclei in human lung squamous cell carcinoma: a useful tool for automatic analysis?","authors":"Ana Vitoria Ferreira Dos Santos, Renan Gabriel da Silva Ferreira, Fernanda das Chagas Angelo Mendes Tenorio, Carina Scanoni Maia, Valdemiro Amaro da Silva Junior, Romildo de Albuquerque Nogueira, Bruno Mendes Tenorio","doi":"10.1007/s00418-024-02336-3","DOIUrl":"https://doi.org/10.1007/s00418-024-02336-3","url":null,"abstract":"<p><p>Lung cancer is the leading cause of cancer-related death. The use of computational methods to quantify changes that are not perceptible to the human eye is increasing in digital pathology imaging and has quickly improved detection rates at a low cost. Therefore, the present study aims to use complex computational shape markers as tools for automated analysis of the spatial distribution of cells in microscopy images of squamous cell lung carcinoma (SqCC). Photomicrographs from pathology glass slides in the LC25000 dataset were used in this study. Compared with those of the control, the fractal dimension (28%) and lacunarity (41%) of the cell nuclei changed in SqCC. The multifractal analysis revealed a significant difference in parameters Dq, α, and f(α) for all values of q (-10 to + 10), with a greater increase for more positive q values. The values at q + 10 increased by 34% for Dq, 36% for α, and 53% for f(α) in the SqCC images. The circularity, area, and perimeter also changed in the SqCC images. However, the parameters of aspect ratio, roundness, and solidity did not significantly differ between SqCC and benign tissue. The complex shape markers with the greatest changes in this study were the f(α) values for multifractality (53%) and lacunarity (41%). In conclusion, automated quantification of the spatial distribution of cell nuclei can be a fast, low-cost tool for evaluating the microscopic characteristics of SqCC; therefore, complex shape markers could be useful tools for software and artificial intelligence to detect lung carcinoma.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":"163 1","pages":"1"},"PeriodicalIF":2.1,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142582730","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Effects of ∆-9 tetrahydrocannabinol on the small intestine altered by high fructose diet: A Histopathological study.","authors":"Basak Isildar, Alisa Bahar Beydogan, Ece Koyuturk, Zeynep Mine Coskun Yazici, Meral Koyuturk, Sema Bolkent","doi":"10.1007/s00418-024-02311-y","DOIUrl":"10.1007/s00418-024-02311-y","url":null,"abstract":"<p><p>The consumption of fructose is increasing day by day. Understanding the impact of increasing fructose consumption on the small intestine is crucial since the small intestine processes fructose into glucose. ∆9-Tetrahydrocannabinol (THC), a key cannabinoid, interacts with CB1 and CB2 receptors in the gastrointestinal tract, potentially mitigating inflammation. Therefore, this study aimed to investigate the effects of the high-fructose diet (HFD) on the jejunum of rats and the role of THC consumption in reversing these effects. Experiments were conducted on Sprague-Dawley rats, with the experimental groups as follows: control (C), HFD, THC, and HFD + THC. The HFD group received a 10% fructose solution in drinking water for 12 weeks. THC groups were administered 1.5 mg/kg/day of THC intraperitoneally for the last four weeks. Following sacrification, the jejunum was evaluated for mucus secretion capacity. IL-6, JNK, CB2 and PCNA expressions were assessed through immunohistochemical analysis and the ultrastructural alterations via transmission electron microscopy. The results showed that fructose consumption did not cause weight gain but triggered inflammation in the jejunum, disrupted the cell proliferation balance, and increased mucus secretion in rats. Conversely, THC treatment displayed suppressed inflammation and improved cell proliferation balance caused by HFD. Ultrastructural examinations showed that the zonula occludens structures deteriorated in the HFD group, along with desmosome shrinkage. Mitochondria were found to be increased due to THC application following HFD. In conclusion, the findings of this research reveal the therapeutic potential of THC in reversing HFD-related alterations and provide valuable insights for clinical application.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":" ","pages":"363-372"},"PeriodicalIF":2.1,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11393283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141897326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"HOXC10 promotes hypertrophic scar fibroblast fibrosis through the regulation of STMN2 and the TGF-β/Smad signaling pathway.","authors":"Xin Zhou, Song Lin","doi":"10.1007/s00418-024-02317-6","DOIUrl":"10.1007/s00418-024-02317-6","url":null,"abstract":"<p><p>The pathophysiology of hypertrophic scar (HS) shares similarities with cancer. HOXC10, a gene significantly involved in cancer development, exhibits higher expression levels in HS than in normal skin (NS), suggesting its potential role in HS regulation. And the precise functions and mechanisms by which HOXC10 influences HS require further clarification. Gene and protein expressions were analyzed using raeal-time quantitative polymerase chain reaction (RT-qPCR) and western blot techniques. Cell proliferation and migration were evaluated using EdU proliferation assays, CCK-8 assays, scratch assays, and Transwell assays. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were conducted to investigate the interactions between HOXC10 and STMN2. HOXC10 and STMN2 expression levels were significantly higher in HS tissues compared with NS tissues. Silencing HOXC10 led to decreased activation, proliferation, migration, and fibrosis in hypertrophic scar fibroblasts (HSFs). Our findings also indicate that HOXC10 directly targets STMN2. The promotional effects of HOXC10 knockdown on HSF activation, proliferation, migration, and fibrosis were reversed by STMN2 overexpression. We further demonstrated that HOXC10 regulates HSF activity through the TGF-β/Smad signaling pathway. HOXC10 induces the activation and fibrosis of HSFs by promoting the transcriptional activation of STMN2 and engaging the TGF-β/Smad signaling pathway. This study suggests that HOXC10 could be a promising target for developing treatments for HS.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":" ","pages":"403-413"},"PeriodicalIF":2.1,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141995687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Distribution and morphologic characterization of telocytes in rat ovary and uterus: insights from ultrastructural and immunohistochemical analysis.","authors":"Merjem Purelku, Hakan Sahin, Gozde Erkanli Senturk, Gamze Tanriverdi","doi":"10.1007/s00418-024-02313-w","DOIUrl":"10.1007/s00418-024-02313-w","url":null,"abstract":"<p><p>Telocytes (TCs) are characterized by a small oval-shaped cell body with long prolongations that are called telopods (Tps). PDGFR-β and c-kit markers may assist for the immunohistochemical identification of TCs; however, by these means they cannot be identified with absolute specificity. Transmission electron microscopy (TEM) is considered as a gold standard method for TCs observation. Studies on TCs in the female reproductive system are limited, and there is a lack of awareness regarding TCs in rat ovaries. We aimed to demonstrate the existence and morphology of TCs in rat ovaries, alongside previously studied TCs in rat uteri. Thus, ovaries and uteri from young adult Sprague-Dawley female rats (n = 8) with regular estrous cycles were collected. Then, left ovaries and uteri were proccessed for TEM analysis, while the right ones were used for immunohistochemistry. As a result, TCs were seen throughout the rat's ovarian stroma with their characteristic cell bodies, Tps, podomes (Pds) and podomers (Pdms). Tps were situated within the thecal layer of the follicles, surrounding the corpus luteum and blood vessels. Ovarian TCs were recognized to have relationship with other TCs/stromal cells. Subsequently, TCs were seen in stroma of endometrium with surrounding blood vessels and uterine glands, myometrium and perimetrium in rat uteri. There was also no statistical significance between the number of c-kit+ and PDGFR-β+ telocyte-like cells in both rat ovarian (p = 0.137) and endometrial stroma (p = 0.450). Further investigation of the roles and functions of TCs in the female reproductive system is needed.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":" ","pages":"373-384"},"PeriodicalIF":2.1,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11393091/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792348","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jihye Kim, Jee Hyun Kim, Younghoon Kim, Jooyoung Lee, Hyun Jung Lee, Seong-Joon Koh, Jong Pil Im, Joo Sung Kim
{"title":"iRhom2 deficiency reduces sepsis-induced mortality associated with the attenuation of lung macrophages in mice.","authors":"Jihye Kim, Jee Hyun Kim, Younghoon Kim, Jooyoung Lee, Hyun Jung Lee, Seong-Joon Koh, Jong Pil Im, Joo Sung Kim","doi":"10.1007/s00418-024-02318-5","DOIUrl":"10.1007/s00418-024-02318-5","url":null,"abstract":"<p><p>Sepsis has a high mortality rate and leads to multi-organ failure, including lung injury. Inactive rhomboid protease family protein (iRhom2) has been identified as accountable for the release of TNF-α, a crucial mediator in the development of sepsis. This study aimed to evaluate the role of iRhom2 in sepsis and sepsis-induced acute lung injury (ALI). TNF-α and IL-6 secretion in vitro by peritoneal macrophages from wild-type (WT) and iRhom2 knoukout (KO) mice was assessed by enzyme-linked immunosorbent assay. Cecal ligation and puncture (CLP)-induced murine sepsis model was used for in vivo experiments. To evaluate the role of iRhom2 deficiency on survival during sepsis, both WT and iRhom2 KO mice were monitored for 8 consecutive days following the CLP. For histologic and biochemical examination, the mice were killed 18 h after CLP. iRhom2 deficiency improved the survival of mice after CLP. iRhom2 deficiency decreased CD68+ macrophage infiltration in lung tissues. Multiplex immunohistochemistry revealed that the proportion of Ki-67+ CD68+ macrophages was significantly lower in iRhom2 KO mice than that in WT mice after CLP. Moreover, CLP-induced release of TNF-α and IL-6 in the serum were significantly inhibited by iRhom2 deficiency. iRhom2 deficiency reduced NF-kB p65 and IκBα phosphorylation after CLP. iRhom2 deficiency reduces sepsis-related mortality associated with attenuated macrophage infiltration and proliferation in early lung injury. iRhom2 may play a pivotal role in the pathogenesis of sepsis and early stage of sepsis-induced ALI. Thus, iRhom2 may be a potential therapeutic target for the management of sepsis and sepsis-induced ALI.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":" ","pages":"415-428"},"PeriodicalIF":2.1,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11393161/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141971016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Maria Eugenia Gulino, Paloma Ordóñez-Morán, Yashwant R Mahida
{"title":"Establishment of a 3D organoid culture model for the investigation of adult slow-cycling putative intestinal stem cells.","authors":"Maria Eugenia Gulino, Paloma Ordóñez-Morán, Yashwant R Mahida","doi":"10.1007/s00418-024-02312-x","DOIUrl":"10.1007/s00418-024-02312-x","url":null,"abstract":"<p><p>The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24 h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.</p>","PeriodicalId":13107,"journal":{"name":"Histochemistry and Cell Biology","volume":" ","pages":"351-362"},"PeriodicalIF":2.1,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141787915","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}